Transcript PowerPoint Presentation - AGRI-MIS

Gibberellins
GAs
Gibberellins (GAs)
a class of plant hormones
affect several important plant processes
eg., seed germination
stem elongation
flowering
male sterility
Gibberellins
1926
Japanese scientist
Gibberella fujikuroi
gibberellin A (terpenoid cpd)
1954, 1955
US and UK scientists
1958
GA1 in higher plant
GAx
1987
synthesis/metabolism
Gibberellins
1991
84 GAs
1995
89 GAs
64 plants, 12 fungi
13 both
1996
more than 100 / 136
1997
genes being cloned
Gibberellic acid (GA3)
End metabolic product in fungi
Plant GA20
GA5
GA3
Commercial
High activity
Slow degradation
Similar to GA1
additional double bond
Gibberellins
GA4 GA7
nonpolar, slowly diffuse
GA9 GA12
precursor
GA29 GA34
deactivated form
Different tissues
Different forms of GA
Gibberellins
fungi
algae
bacteria
moss
fern
gymnosperm
angiosperm
Gibberellins
growing, differentiated tissues
young, developing, expanding leaves
developing seeds/fruit
Gibberellins
elongated internode/petiole
shoot/stem apex
root cap/tip
xylem sap
Synthesis and Metabolism
Mevalonic acid pathway
in cytosol
Non mevalonic acid pathway
in plastid
Mevalonic acid pathway
In higher plants
from GA12 aldehyde
Early 13-hydroxylation pathway
(GA1)
Non 13-hydroxylation pathway
(GA4)
with GA20oxidase genes:
pathway shifted
GA4 increased / GA1 decreased
GA12 aldehyde: precursor of GA derivatives
by oxidation (C20) and hydroxylation (C13 C3 C2)
Vegetative tissue: conserved synthetic pathway
13-OH pathway to GA20 (C19-GA)
then 3b-OH to GA1
except: arabidopsis and cucumber
non 13-OH pathway to GA4
Reproductive tissue/seed: various pathways
different forms of GA
From
mevalonic acid (6C)
GGPP (20C-linear cpd)
ent kaurene (1st specific cpd)
GA12 aldehyde (first GA)
GAx
Gibberellins
Isoprene (5C) as basic unit
ent-Gibberellane skeleton
tetracyclic diterpenoid cpd
2 main types:
C20-GA and C19-GA
GA derivatives by modification of 4 rings
* C20 oxidation: CH3 CH2OH CHO COOH
* Hydroxylation at C2 C3 and C13:
number, position
stoichiometry
* Loss of C20 (C20 to C19 GA)
GA inactivation
* 2b-OH: GA20
GA1
* C20 oxidation
GA29
GA8
to COOH
GA inactivation
* Conjugation by glucose
Glycosylation:
inactive, storage and transport
Glucose via COOH: GA glycoside
Glucose via OH: GA glycosyl ether
GA synthesis mutants
Pea na mutant: dwarf
ent-kaurene
Pea le mutant: dwarf
GA12 aldehyde
exogenous GA1
tall
exogenous GA20
no response
cloned Le gene: 3b hydroxylase
GA20
GA1
Considering 2 loci
na Le
normal ent-kaurene
Na le
normal GA20
Grafting
1.
na Le
Na le
2.
Na le
Na Le
Conclusion?
scion
stock
tall
scion
stock
dwarf
GA mechanism in elongation
Unlike auxin (acidification)
Increase wall extensibility
Decrease minimum force
for wall extension
GA mechanism in elongation
By (may)
decrease Ca concentration in the wall
increase Ca uptake into the cell
reduce crosslinking of lignin-related cpd
(via peroxidase)
GA mechanism in germination
Activate transcription of
a amylase gene
In scutellum and aleurone
GA detection and assay
Bioassay
Easy but not specific
Fractionation
Plant response
Lettuce hypocotyls elongation
Microdrop/dwarf rice
a amylase production
GC-MS
Solvent extraction
Chromatography (polarity)
GC (boiling point)
MS (mass)
Identification and quantification
High sensitivity and more specific
GA inhibitors
Inhibit ent-kaurene synthesis
AMO1618
Cycocel
Inhibit ent-kaurene oxidation
Paclobutrazol
Uniconazol
Ancymidol
Tetcyclasis
Inhibit later steps by dioxygenases
Bx-1112
LAB1988999
Hormone
Responses
Perception: receptor
Signal transduction:
second messenger (cAMP, cGMP)
G protein
Ca-Calmodulin
enzyme
transcription factor
At last step
Gene expression
Specific region in promoter
cis element
DNA-binding protein
transcription factor
GA studies
Exogenous GA / GA inhibitor
GA mutant
Gene identification / Gene cloning
Gene expression / Transformation
GA synthesis
Enzyme: gene product of multigene family
Each gene with specific pattern of expression
AtGA20ox1: shoot growth
AtGA20ox2: inflorescence development
AtGA20ox3: early seedling development
At later steps of synthetic pathway
Genes controlled by GA, light and daylength
GA: inhibit transcription of GA20oxidase
(GA19 to GA20)
inhibit 3b hydroxylase
promote 2b hydroxylase
Negative
Light:
feedback:
promote
reduce
conversion
production
GA1
of active
to inactive
GA20GA8
and GA1
Daylength
(LD):
floralofinitiation
activates
activity
reducing
shoot GA20oxidase
elongation
GA53
to
GA44
GA19
to
GA20
Pea, Pisum sativum
In de-etiolated pea seedling, exposed to
red, blue, far red, all reduce GA1 level
Lettuce: Lactuca sativa seed germination
Red light:
activates LsGA3ox1 expression
GA1 increase
Far-red light: inhibits LsGA3ox1
Auxin: promote GA1 production
inhibit deactivation steps to GA29 and GA8
GA synthetic mutants
Arabidopsis:
seed germination assay
5 complementation groups (56 lines)
ga1 ga2 ga3 ga4 and ga5
all recessive, dwarf, and male sterile
ga1 and ga2 reversed by ent-kaurene
ga3 reversed by ent-kaurenal
Genes
GA1 kaurene synthase (ent-CDP synthase)
GA3 Cyt P450-dependent monooxygenase
GA4 3b hydroxylase
GA5 GA20oxidase
Pea (sln)
decrease 2b hydroxylase activity
increase active GA
tall plant with light green leaves
Signal transduction mutants
Stature mutants
Decreased response to GA
Increased response to GA
Decreased signaling mutants
Dwarf
Complete phenocopy of
GA-deficient mutants
No response to exogenous GA
Decreased signaling mutants
Partially / fully dominant
Arabidopsis gai
Maize D8 D9
Wheat Rht1 Rht2 Rht3
Negative regulators
Arabidopsis gai mutant
Dwarf
Higher level of active GA
and GA20oxidase
Semidominant
Arabidopsis gai mutant
gai1-1
51 bp inframe deletion
loss of 17 amino acid
constitutive repressor
Arabidopsis gai mutant
intragenic suppressor of gai
loss of function allele
WT phenotype
Maize D8 mutant
Dwarf
Higher level of active GA
6 dominant alleles
with different severity
Wheat Rht mutant
8 dominant alleles with different severity
Dwarf: prevent lodging
Wheat + N fertilizer: increase yield
increase height
Norin10: dwarf line
2 mutated loci: Rht1 or Rht-B1b (chrs 4B)
Rht2 or Rht-D1b (chrs 4D)
All genes cloned:
deduced amino acid sequence
GAI / Rht / d8 homologs
Conserved domains I and II in N terminal
gai mutant:
deletion in domain I
D8 / Rht: mutation in domain I and/or II
*N terminal essential for GA response*
Increased signal transduction mutants
Similar to WT + GA
Tall by elongated internodes
Arabidopsis
Barley
Rice
Tomato
Pea
spy rga
sln spy
slr
pro
la crys
Recessive / Negative regulators
Increased signal transduction mutants
Arabidopsis rga
Identified by suppression analysis of ga1-3
New mutant: taller
ga1-3 <
ga1-3* < WT
new locus: repressor of ga1-3 (rga)
Increased signal transduction mutants
rga: recessive (deletion mutation)
increase stem elongation
reverse ga1-3 delayed flowering time
no effect on GA biosynthesis
RGA: negative regulator
Gene: 82% homology to GAI
especially in N region
Original gai mutant: gain of function
Loss of function allele of GAI ?
Phenotype: normal
Increase paclobutrazol resistance
Low GA = normal height
At least two components in
Arabidopsis GA signaling pathway
GAI and RGA
homopolymeric Serine / Threonine residue
leucine heptad for protein-protein interaction
putative nuclear localizing signal
Barley sln
slender mutant
recessive
long internodes and narrow leaves
male sterile
increase a-amylase w/o GA
low endogenous GA
resistant to GA synthesis inhibitors
negative regulator
sln x dwarf mutant = sln phenotype
SLN = GAI/RGA homolog
Dominant allele of SLN mutant
Mutation in N terminal
Dwarf barley
Rice slr
slender rice
recessive
phenocopy of barley sln
1 bp deletion in NLS domain
(nuclear localization signal )
Rice slr
frame shift mutation
stop codon
truncated protein
SLR gene = SLN homolog
Modified SLR:
17 aa deletion in DELLA domain
Transformation: dwarf rice
GA signal component
Dicot / Monocot
GAI RGA Rht d8 SLN SLR
Putative transcription repressor
Arabidopsis spy
spindly mutant, recessive
paclobutrazol-resistant
long hypocotyls
light green leaves
early flowering
spy ga1-2 = spy phenotypes
spy gai
= spy phenotypes
Arabidopsis spy
SPY gene product:
O-GlcNAc transferase
Signaling molecule
Involved in protein-protein interaction
Negative regulator
Before responses
Expression of GA-regulated genes:
Protein-DNA interaction
Transcription factor
cis elements
Transcription factor: GAMyb
Barley: HvGAMyb
Bind specific sequence in
promoter of a-amylase gene
Increase gene expression
Overexpression of HvGAMyb gene
= GA treatment
Arabidopsis: GAMyb-like genes
AtMyb33 AtMyb65 AtMyb101
Functional homologs of barley GAMyb
Transform barley aleurone with AtMyb33
Activate a-amylase production
Arabidopsis:
facultative LD plants
Transfer plants from SD to LD
11x increase of GA1
3x
increase of GA4
increase AtMyb33 expression
in shoot apex
shoot apex transition to flowering
Potential target for AtMyb
LFY promoter
LEAFY: meristem-identity gene
Evidence AtMyb binding
to a specific 8-bp sequence
in LFY promoter
cis elements
specific regions in promoter
transcription factor binding site
identified by deletion or
site specific mutagenesis:
gene expression after promoter modification
Conserved sequences among
GA-regulated genes
- amylase box:
TATCCAT
- GARE:
TAACAA/GA
- Pyrimidine box:
C/TCTTTTAC/T
GA and a-amylase production
Perception at membrane receptors
Increase intracellular Ca
Decrease intracellular pH
Increase [CaM]
Increase cGMP
Increase GAMyb transcription
Increase a-amylase activity
Some protein phosphorylation